The present invention relates in general to the preparation of derivatized polynucleotides. In particular, the present invention relates to the derivatization of the 3' end of synthetic polynucleotides.
The attachment of a functional group to a nucleic acids permits its detection and quantitation. In a hybridization assays a labelled nucleic acid probe is used to search a sample for a target nucleic acid which has a complementary nucleotide sequence. The target is then immobilized by hybridization to a support-bound nucleic acid probe to form a "sandwich". Such a sandwich is detectable as the amount of label bound to the support. Functionalization techniques may be employed both to attach a reactive group for binding the nucleic acid to a reporter group and to bind the nucleic acid to a reactive group on a support. Thus, methods for derivatizing nucleic acids are particularly useful for preparing materials for hybridization assays.
One approach to labelling a probe for use in hybridization assays involves binding a radioisotope (e.g., .sup.32 P, .sup.3 H, or .sup.125 I) to the probe. However, difficulties inherent in the two common methods of detecting radioactive labels limit the usefulness of its approach. One of the common detection techniques, called, autoradiography, is a timeconsuming procedure which relies upon reduction of silver ions to form silver grains in a photographic emulsion. Scintillation counting, the other common detection technique, requires expensive equipment and a certain amount of delay as well. Furthermore, radioisotopes require special handling for safety reasons. Some radioactive isotopes, such as .sup.125 I, have relatively short shelf-lives, which further limit their usefulness in a clinical diagnostic setting.
In non-radioactive labelling systems, a probe is "labelled" with a reporter group and a signal is associated with the reporter group to enable detection. A reporter is an agent which is used to indicate the presence or location of the probe. The signal itself, which is directly perceptible, may be generated by a separate or separable signal molecule. A label is properly a type of reporter which incorporates a signal molecule.
One approach to the attachment of labels to probes is described in Ward, et al., European Patent Application No. 63,879. Ward discloses the preparation of probes having a biotin reporter molecule covalently attached to a purine or pyrimidine ring. Selected biotinylated purines and pyrimidines are then directly incorporated within the phosphodiester backbone of the nucleic acid of the probe by enzymatic means. However, enzymatic techniques are costly and difficult to perform.
Other approaches link a label to a probe by way of a protein. Single-stranded polio virus RNA is naturally linked to a protein which may be reacted with the N-hydroxysuccinimidyl ester of biotin to obtain an RNA probe having a biotinylated reporter group detectable by specific attachment of avidin-coated spheres. Richards, et al., Proc. Natl. Acad. Sci. (U.S.A.), 76: 676-680 (1979). Similarly, biotin-labelled cytochrome c may be coupled to RNA by reaction in the presence of formaldehyde and thereafter labelled with avidin-coated spheres. Manning, et al., Chromosoma (Berl.), 53: 107-117 (1975). Nevertheless, because not all nucleic acids are naturally linked to proteins and because the location and amount of cytochrome c binding to a nucleic acid is not readily predictable, it would be desirable to have a chemical synthetic technique for end-labelling.
In one chemical synthetic technique, nucleic acids are converted to 3'-aldehydes by oxidation and condensed with alkyldiamines or polyamines to provide a reporter group for the attachment of biotin. Broker, et al., Nucleic Acids Res., 5: 363-384 (1978). Similarly, aldehydes generated by the periodate oxidation of nucleic acids may be used to couple fluorescent labels to the nucleic acids. Bauman, et al., J. Histochem.Cytochem., 29: 227-237 (1981). However, it would be desirable to have a technique for attaching reporter groups to polynucleotides bound to a support in an automated process for nucleic acid synthesis.
In yet another approach to 5' labelling, biotin is converted to 2-(biotinylamido)ethanol and condensed to a phosphorylated, polymer-supported nucleotide. The condensation of the aminoethanol derivative of biotin to the 5' hydroxyl group of a ribose ring gives a stable phosphodiester bond upon deprotection of the nucleotide. Kempe, et al., Nucleic Acids Res., 13: 45-57 (1985). Nevertheless, because specific reporter groups are attached, this approach does not teach preparation of an oligonucleotide with a generally reactive functionality which may later be used to attach a variety of desired reporter groups.
Nucleotides in solution have been amine-functionalized by condensation with protected 6-amino-1-hexanol phosphate. Barker, et al., J. Biol. Chem., 22: 7135-7147 (1972). However, these procedures are difficult to perform and have not been integrated with solid-phase synthesis.
In another approach to binding nucleotides to supports for the purification of nucleases by affinity chromatography, single nucleotides, 3'-derivatized with p-aminophenol are attached to a gel matrix by a linker. The linker is formed by attaching 3,3'-diaminodipropylamine to the matrix using cyanogen bromide and azide. The resulting amine-functionalized gel is treated with succinic anhydride and then coupled to the amine-functionalized nucleotide with a carbodiimide. Cuatrecasas, J. Biol. Chem., 12: 3059-3065 (1978). Nevertheless, the manufacture of the amine-functionalized nucleotide itself is performed in solution by tedious procedures. See e.g., Barker et al, J. Biol. Chem., 22, 7135-7147 (1972).
Therefore, there is a need for a method and composition for the generic attachment of reporter groups to polynucleotides undergoing solid phase synthesis.